Elucidation Of Cellular Damage During Exposure To Oxidat

Heart, Lung, And Blood Institute

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Abstract

Research in the Section on Enzymes in the Laboratory of Biochemistry, NHLBI, is directed toward elucidation of basic mechanisms involved in the production of cellular damage during exposure to oxidative stress, and the contributions of such damage to aging and disease. To this end, our current research involves studies in the following areas of exploration: (a) Antioxidant role of cyclic oxidation and reduction of methionine residues of proteins. Reactive oxygen-mediated oxidation of methionine residues of proteins leads to formation of a racemic mixture of the R- and S-stereo isomers of methionine sulfoxide. Most biological systems contain two forms of methionine sulfoxide reductase: one that contains a cysteine moiety at the catalytic site and is specific for the reduction of the S- isomer of the sulfoxide back to methionine, and an another that contains selenocysteine at the catalytic site and is specific for reduction of the R-isomer. To investigate the effects of selenium on the antioxidant activity of methionine interconversion, mice were grown on a selenium deficient diet. Selenium deficiency led to large increases in the tissue levels of oxidized protein, as measured by methionine sulfoxide content and the protein carbonyl content. Efforts to clarify the role of selenium deficiency in protein oxidation and the effect of over-expression of methionine sulfoxide reductase in mice are in progress. (b) Inflammation-induced oxidation of methionine residues of proteins. The biosynthesis of hypochlorous acid by neutrophils and macrophages represents a major mechanism for antibacterial action in mammals. Because methionine residues of proteins are particularly sensitive to oxidation by hypochlorous acid, we carried out studies to elucidate the mechanisms involved. In view of the fact that the reactions of hypochlorous acid might be influenced by the kind of buffer used in in vitro experiments and the fact that most studies to date have been carried out in non-physiological buffer systems, we carried out studies using the physiological bicarbonate buffer. Results of these studies indicate that hypochlorous acid reacts readily with components of the bicarbonate buffer to form an intermediate which is also able to oxidize methionine residues of proteins. Further studies will be directed toward determination of the structure of this intermediate and to identify the products formed in the hypochlorous-bicarbonate dependent oxidation of methionine residues of proteins. (c) Role of apoptosis in aging. When animals reach maturity, the size of various tissues is fixed. Therefore, oxidative damage to cells in these tissues will lead to loss of tissue function unless the damaged cells are removed and then replaced by good cells. Significantly, at low concentrations, reactive oxygen species are able to activate cell signaling pathways leading to the removal of damaged cells by apoptosis and also pathways involved in the activation of cell replication. The possibility that these signaling processes provide a mechanism for maintaining the integrity of mammalian tissues is supported by results of preliminary studies showing the inhibition of apoptosis in cultured leukemia NB4 cells, followed by exposure of the cells to oxidative stress, leads to accumulation of oxidatively damaged protein in these cells. (d) Effect of ribonucleic acid (RNA) oxidation on translational efficiency and accuracy. Oxidative modification of ribonucleic acid is associated with several neurological disorders. To study the effect of ribonucleic acid oxidation on its translational efficacy, ribonucleic acid encoding the luciferase gene was subjected to oxidation by hydrogen peroxide and its ability to produce luciferase when incubated in reticulocyte lysate was examined. Results of preliminary experiments indicate that activity of the luciferase protein translated from oxidized ribonucleic acid is considerably lower than that of normal luciferase preparations. Further studies will be made to determine if the modified form of luciferase generated by the oxidized ribonucleic acid reflects miss-incorporation of amino acids. (e) Regulation of caspase-12 transcription. We showed earlier that high concentrations of manganese induces apoptosis in NIH 3T3 cells by a caspase-12-mediated mechanism. To elucidate basic mechanisms involved in the regulation of caspase-12 gene transcription, we isolated and sequenced two fragments of the 5' flanking region and the 5' untranslated region (5' UTR) of the mouse caspase-12 promoter and cloned them into the pGL3 promoter-less vector upstream of the luciferase gene. We also isolated the caspase-12 3'UTR which is downstream of the caspase-12 gene and inserted into pGL3 downstream from the luciferase gene which is under control of the SV40 promoter. The effects of the 5' UTR and 3' UTR constructs were monitored in NIH 3T3 cells which were grown in the presence and absence of 10% serum. Results of the studies with the 5'UTR construct suggest that serum contains factors that are required for translation of the luciferase mRNA and for down regulation of the caspase-12 gene. In contrast, studies with the 3'UTR construct showed that serum contains a factor that is required for the down regulation of the luciferase gene. Together, these findings represent the first characterization of the caspase 5' and 3' UTR region of the caspase 12 gene and should facilitate a better understanding of transcriptional and translational control.